Zadeh Cameron O, Huggins Jonah R, Sarmah Deepraj, Westbury Baylee C, Interiano William R, Jordan Micah C, Phillips S Ashley, Dodd William B, Meredith Wesley O, Harold Nicholas J, Erdem Cemal, Birtwistle Marc R
Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina 29634, United States.
Department of Bioengineering, Clemson University, Clemson, South Carolina 29634, United States.
ACS Omega. 2022 Aug 11;7(33):28912-28923. doi: 10.1021/acsomega.2c02201. eCollection 2022 Aug 23.
Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed Microwestern requires a piezoelectric pipetting instrument, which is not available for many labs. Here, we report the Mesowestern blot, which uses a 3D-printable gel casting mold to enable high-throughput Western blotting without piezoelectric pipetting and is compatible with the standard sample preparation and small (∼1 μL) sample sizes. The main tradeoffs are reduced molecular weight resolution and higher sample-to-sample CV, making it suitable for qualitative screening applications. The casted polyacrylamide gel contains 336, ∼0.5 μL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution profiles. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder and cell lysate (RIPA buffer) demonstrate that the protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semidry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is at least 32-fold, and at least ∼500 attomols of β-actin can be detected (∼29 ng of total protein from mammalian cell lysates: ∼100-300 cells). Because the gel mold is 3D-printable, users with access to additive manufacturing cores have significant design freedom for custom layouts. We expect that the technique could be easily adopted by any typical cell and molecular biology laboratory already performing Western blots.
蛋白质免疫印迹法是一种广泛应用于蛋白质分子量解析分析及其翻译后修饰的技术，但标准平板凝胶装置的高通量应用却很少见。先前开发的微孔蛋白质免疫印迹法需要一种压电移液仪器，许多实验室都没有这种仪器。在此，我们报告了中孔蛋白质免疫印迹法，该方法使用一种可3D打印的凝胶浇铸模具，无需压电移液即可实现高通量蛋白质免疫印迹，并且与标准样品制备和小体积（约1 μL）样品兼容。主要的权衡之处在于分子量分辨率降低和样品间变异系数较高，这使得它适用于定性筛选应用。浇铸的聚丙烯酰胺凝胶在标准微孔板尺寸范围内包含336个约0.5 μL可通过微量移液器加样的样品孔。可以改变聚丙烯酰胺的百分比以改变分子量分辨率曲线。使用红外荧光分子量蛋白标准品和细胞裂解液（RIPA缓冲液）进行的概念验证实验表明，中孔蛋白质免疫印迹凝胶中加载的蛋白质适用于标准的蛋白质免疫印迹步骤。中孔蛋白质免疫印迹法与传统蛋白质免疫印迹法的主要区别在于使用的是半干水平凝胶电泳而非浸入式垂直凝胶电泳。检测的线性范围至少为32倍，并且至少可以检测到约500阿托摩尔的β-肌动蛋白（来自哺乳动物细胞裂解液的约29 ng总蛋白：约100 - 300个细胞）。由于凝胶模具是可3D打印的，能够使用增材制造核心设备的用户在定制布局方面有很大的设计自由度。我们预计，任何已经在进行蛋白质免疫印迹的典型细胞和分子生物学实验室都可以轻松采用该技术。
